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The mucosal barrier is a well-armed opponent against the microbial world that works to limit entry of pathogenic bacteria, while maintaining a level of tolerance towards the resident microbiota. This paradoxical function is carried out by three distinct barriers i.e. physical, innate, and adaptive. Microvilli, a component of the physical barrier, while noted for their capacity to take up nutrients, have been overlooked as playing a role in the prevention of bacterial entry. We report that microvilli generate an electrostatic barrier that directs pathogen binding based on their zeta potential (proxy of surface charge). By knocking out microvilli in an intestinal cell line we were able to probe how bacterial adherence/uptake was affected by the absence of microvilli. This electrostatic barrier may have important functional consequences at the surface of the follicle- associate epithelium (e.g. Peyer's patch), where interdispersed between adjacent enterocytes reside M cells, which lack microvilli. M cells are a feature of the adaptive barrier, and are known for their ability to transcytose luminal antigens in the small intestine. However, their role in the large intestine has been neglected. We describe the induction of M cells in the colonic epithelium during intestinal inflammation, which seems to be reliant on the pro-inflammatory cytokine TNFα, as treatment with anti-TNFα largely blocked M cell induction. This was confirmed using a novel reporter mouse that was engineered to express DsRed in both neutrophils and M cells. Thus, M cells may play a greater role during inflammation. Furthermore, M cells also mediate adaptive immune response through associated B lymphocytes. The follicle of the Peyer's patch is heavily populated with B lymphocytes that help to generate secretory IgA. Large portions of these B cells are of the B1 subset, known for their capacity to stimulate T cell independent antibody response in the presences of polymeric antigens. We describe the design of a T cell independent antigen using polymeric flagellin as a vaccine backbone to stimulate the T cell independent B cells. We incorporated the envelope protein from dengue virus 2 into the D3 domain of flagellin and these hybrid filaments were able to produce a humoral response in both T cell dependent and independent models.

M cells are specialized epithelial cells, which assist immune surveillance by transcytosis of particles and antigens to underlying lymphoid tissues. So far, three M cell phenotypes have been identified in airways and intestines. However, the mechanism of M cell differentiation is poorly understood, as well as the relationships between different M cell subtypes. To better understand these questions, we treated human (Caco-2BBe) and rodent (IEC-6) intestinal epithelial cell lines with lymphotoxin beta receptor (LTbetaR) and TNF receptor (TNFR) agonists. Treated cells were then studied for regulation of genes previously shown to associate with M cell and Follicle-Associated Epithelium (FAE). Moreover, we used a reporter transgenic mouse strain to follow the development of M cell subsets in vivo, and examined the de novo induction of M cells by cholera toxin (CT).We found that LTbetaR and TNFR agonists can induce transcription of FAE specific genes (Ccl20 and Lamb3) as well as rodent M cell specific genes such as Sgne-1/Scg5, Cldn4, and Gp2 in Caco2-BBe cells and IEC-6 cells. These cytokines have distinct but complementary effects; TNFR agonists induced FAE specific genes, while the LTbetaR agonist induces M cell specific genes. The combination of these cytokines showed additive induction of the FAE-associated Ccl20, Lamb3 and a surprisingly the induction of CD137/Tnfrsf9. Functionally, cytokine treatment led to both the reorganization of microvilli as well as Claudin-4 redistribution. Moreover, with the help of the reporter transgenic mouse model, we have found that M cells overlying intestinal Peyer's patches (PP) and Nasal Associated Lymphoid Tissue (NALT) have distinct developmental origins, yet show convergent phenotypes, such as expression of a PGRP-S reporter in vivo. By contrast, though PP and Villous M cells are derived from intestinal crypt stem cells, their phenotypes are clearly distinct. Thus, PP M cells form an intimate M cell-dendritic cell functional unit while Villous M cells do not engage with underlying DC. Though B lymphocytes are thought to play a critical role in M cell function by forming a basolateral pocket and possibly signaling through CD137, initial commitment to M cell lineages is B lymphocyte and CD137 independent. In addition, CT can causes rapid induction of new M cells in the airway and intestines without cell division, suggesting transdifferentiation from mature epithelial cells. CT-induced M cells show the local M cell phenotype without evidence for mixed phenotypes. Finally, M cell phenotypes display distinct capabilities; thus, intestinal PP M cells were more efficient than Villous M cells in the uptake of Salmonella. Our studies show that LTbetaR and TNFR agonists induce many features associated with M cell differentiation, and suggest that these agonists may be involved in secondary lymphoid tissue development in vivo. Additionally, we confirmed that the differentiation of M cells in vivo toward strictly defined phenotypic subsets, and is consistent with functional specialization.